The Human phospho kinase array was performed ac cording the protocol of the manufacturer. In this array, 46 capture antibodies are spotted in selleckchem duplicate on nitro cellulose membranes. STAT1, STAT2, STAT3, STAT4, Inhibitors,Modulators,Libraries STAT5a, STAT5a b, STAT5b, STAT6, TOR, Yes and B catenin. In short, cell lysates were incubated with the membrane overnight. Thereafter, the membranes were incubated with a cocktail of biotinylated detection antibodies and streptavidin HRP. Finally, proteins were detected using an ECL chemiluminescent system. To quantify expression levels, the integrated optical density of each spot was measured using ImageJ software. IOD values were corrected for background signal and to compare different membranes levels were normal ized to those of the positive controls on each membrane.

Both the absolute expression levels after radiotherapy as well as the relative levels after radiotherapy Inhibitors,Modulators,Libraries were quantified. Radiosensitivity Clonogenic cell survival assays Cells were irradiated with graded doses at room temperature. After 1. 5 3 weeks, depending on the growth speed of the cell line, cells were stained with 0. 5% crystal violet and colonies with more than 50 cells were counted. Clonogenic survival curves were fitted using the linear quadratic model and the surviving frac tion after 4 Gy was calculated using the and B values obtained from the curve. Kinase inhibition Clonogenic cell survival assays western blot analyses For clonogenic cell survival assays, cells were incubated with the kinase inhibitor for 16 h and then irradiated with 4 Gy.

Thereafter, cells were treated with the kinase inhibitor for 72 h and subse quently cells were incubated in drug free medium. After 1. 5 3 weeks, cells were stained with crystal violet and colonies were counted. Survival fraction after combined treatment with 4 Gy and the kinase inhibitor was calcu lated by correcting Inhibitors,Modulators,Libraries for plating efficiency of the untreated control or by correcting for plating efficiency of cells treated with the inhibitor alone. For western blot analyses, cells were treated with the inhibitor for 16 h followed by irradiation with 4 Gy and harvested 4 Inhibitors,Modulators,Libraries h after radiotherapy or 20 h after kinase treatment. Cells Inhibitors,Modulators,Libraries were lysed in RIPA buffer and protein was quantitated using a standard Bradford absorbance assay. Proteins were separated by SDS PAGE and blotted onto PVDF membrane.

Membranes were incubated with the appropriate primary antibodies followed by incubation with HRP conjugated antibodies. Finally, proteins were detected using chemilumines sellekchem cence. Antibodies against the following antigens were used p p38, pMEK1 2, pMSK1, pSFK, pSTAT6, pSTAT5, pAKT, pERK1 2, and HRP conjugated goat anti rabbit IgG were purchased from Cell Signaling Technology, HRP conjugated goat anti mouse IgG was purchased from Santa Cruz Bio technology, and tubulin was obtained from Calbiochem.